To better understand how proteins execute their designed function, much effort has gone into the determination of their three-dimensional structures. For example, of the 12,000+ protein structures deposited into the Protein Data Bank, nearly 3,300 are characterized as enzymes. These static structures are enlightening; however, the side chains surrounding the active site of an enzyme are not static spectators but are active participants in the choreographed motions that mediate chemical transformation. To fully understand how an enzyme functions at the molecular level, it is crucial to know the structural changes that ensue as it executes its designed function. With this knowledge, researchers will be better poised to rationally engineer proteins and peptides with therapeutic value. To watch a protein as if functions with atomic spatial resolution, we have developed the technique of picosecond time-resolved X-ray crystallography. This technique is based on the pump-probe method where a picosecond laser pulse (pump) triggers a reaction in a protein crystal and a delayed X-ray pulse (probe) takes a "snapshot" of the protein's structure. These experiments require an intense source of X-ray pulses and synchronized tunable laser pulses. The ID09B time-resolved beamline at the European Synchrotron and Radiation Facility (ESRF) in Grenoble, France is still the only beamline in the world capable of recording time-resolved macromolecular structures with 150 picosecond time resolution. Using this source, we have studied the structural changes that accompany ligand migration in myoglobin (Mb). This protein has proven to be a very useful model system for these investigations: mutant forms can be over expressed in E. coli; it forms highly ordered crystals that diffract to atomic resolution; it reversibly binds small ligands such as O2, CO, and NO; and it can be photolyzed with high efficiency. In particular, we have focused on the L29F mutant of myoglobin (Mb), where the leucine (L) in the 29 position is replaced by phenylalanine (F). According to femtosecond time-resolved IR measurements of photolyzed L29F MbCO, the rate of ligand escape from its primary docking site is accelerated at least 1000-fold compared to wild-type MbCO. We have acquired structures of this mutant at time delays spanning from 100 picoseconds to 3 microseconds. The structural rearrangements triggered by ligand dissociation are striking, and involve correlated motion of the heme and numerous side chains. In particular, to accommodate docked CO, the phenylalanine in position 29 is displaced toward the distal histidine, and this sterically strained intermediate rapidly sweeps CO out of the docking site with a significant population ending up in the Xe4 docking site. On the time scale of some tens of nanoseconds, the CO migrates around the heme to the Xe1 docking site. The correlated structural changes are most clearly unveiled in a movie produced by stitching together the complete series of time-resolved structures. We are currently extending these studies to hemoglobin as well as other mutants of myoglobin. The technique of time-resolved X-ray crystallography is still in its infancy, and much work remains to be done to enhance the quality of data acquired, to develop improved tools to analyze the diffraction data, and to develop improved tools to visualize the 3-D data. Dr. Eric Henry (LCP) is collaborating with us to develop new tools to analyze diffraction images and translate them into 3D electron density maps. Dr. Gerhard Hummer (LCP) is running molecular dynamics simulations to help identify the molecular basis for the structural changes that are observed. Our femtosecond time-resolved spectroscopy lab continues to study the photophysics of various chromophores in protein crystals in order to develop more efficient methods for photoactivation. This combination of spectroscopic, crystallographic, and computational tools are paving the way to explore functionally-important structure transitions at an atomistic level, from which a far more meaningful mechanistic description of protein function will be achieved.